PROJECTION IS AN INVERTEBRATE CONNECTIN (TITIN) - ISOLATION FROM CRAYFISH CLAW MUSCLE AND LOCALIZATION IN CRAYFISH CLAW MUSCLE AND INSECT FLIGHT-MUSCLE

A filamentous protein was isolated from crayfish claw muscle. This protein had physiochemical properties very similar to vertebrate skeletal muscle connectin (titin), although its apparent molecular mass (approximately 1200 kDa) was considerably lower than that of connectin (approximately 3000 kDa). Polyclonal as well as monoclonal antibodies against chicken skeletal muscle connection reacted with the 1200 kDa protein from crayfish claw muscle. Conversely, polyclonal antibodies against crayfish 1200 kDa protein cross-reacted with chicken connectin. Circular dichroic spectra indicated the abundance of beta-sheet structure (approximately 60%). Low-angle shadowed images showed filamentous structures (0.2 approximately 0.5-mu-m) by electron microscopy. Proteolysis of the 1200 kDa protein by alpha-chymotrypsin or V8 protease rapidly resulted in formation of 1000 kDa or 1100 and 800 kDa peptides. The amino acid composition was very similar to those of vertebrate connections and of honeybee flight muscle projectin. Based on the molecular weight and amino acid composition, the 1200 kDa protein is regarded to be crayfish projectin. Immunofluorescence and immunoelectron microscopy revealed that crayfish projectin was localized in the A/I junction area and A-band except for its centre region in crayfish claw muscles. Polyclonal antibodies against crayfish claw muscle projectin reacted with 1200 kDa projectin of honeybee and beetle flight muscle. A monoclonal antibody against chicken skeletal muscle connectin also reacted with honeybee and beetle projectin. Immunoelectron microscopic observations revealed that anti-crayfish projectin antibodies bound the connecting filaments linking the Z-line and the thick filaments up to the M-line of honeybee muscle sarcomere. Anti-crayfish projectin antibodies bound the I-band region near the Z-line of beetle flight muscle. It is concluded that the 1200 kDa projectin from crayfish claw muscle is an invertebrate connectin (titin). Recent work with locust flight muscle mini-titin (Nave & Weber, 1990) is in good agreement with the present study, except that the isolated minititin estimated as 600 kDa appears to be a proteolytic product (approximately 1100 kDa) of the parent molecule (approximately 1200 kDa).